Birth defects are among the leading pediatric healthcare issues, yet there are few prevention strategies and the prevalence of birth defects in the US has remained relatively stable for decades, despite folic acid fortification of the food supply. Neural tube defects (NTDs) are among the most common and serious of all birth defects affecting 0.5-1 per 1,000 US newborns, yet their complex etiology remains poorly understood (Wallingford et al., 2013). It is well established that pre-pregnancy supplementation with folic acid can reduce the risk of NTDs, but the underlying mechanism of this beneficial effect is unclear. Approximately 30% of NTDs cannot be prevented by maternal periconceptional folate supplementation. Our preliminary data showed that rare mutations in planar cell polarity (PCP) pathway could increase the risk of NTDs, while mutagens such as polycyclic aromatic hydrocarbons were enriched in the placentas of NTDs fetuses/infants. We hypothesized that NTDs are caused by combinations of rare multiple mutations. Higher mutation rates results in a higher NTDs prevalence. It is possible that folic acid can prevent NTDs by reducing the mutation rate. Furthermore, we hypothesize that paternal supplementation with folic acid can further reduce NTD rates. We have a unique opportunity to evaluate folate supplement effect on mutation rate by leveraging previously collected biological samples from NTD patients and controls from several distinct cohorts. Additionally, we propose to use mouse models to better understand the relationship between folate and mutation rates in vivo. The results of this study will provide an explanation of how folate prevents NTDs, and will reveal novel pathways for intervention on folate-resistant NTDs. Hence, the successful completion of the proposed studies is likely to have substantial impact on our understanding of conditions that affect ~6% of births worldwide and constitute one of the major healthcare concerns for the youngest members of society.